The development of the mammalian kidney requires the inductive interactions of two progenitor tissues, the ureteric bud epithelium and the metanephric mesenchyme. Signals emanating from the ureteric bud induce the mesenchyme to proliferate and differentiate, thus generating most of the epithelial cell types in the nephron. Reciprocally, mesenchyme derived signals induce the ureteric bad epithelium to proliferate and undergo branching morphogenesis to generate the collecting duct system. Although these signals and their receptors have remained elusive, their mechanisms of action are fundamental to understanding the molecular basis of renal cell growth and differentiation. The receptor tyrosine kinase RET is expressed at the tips of the ureteric bud and is essential for ureteric bud outgrowth and branching. Recent work in the PI's lab has demonstrated that the glial cell derived growth factor GDNF, expressed in the metanephric mesenchyme, activates the RET receptor and can stimulate branching morphogenesis in kidney organ cultures. Thus, GDNF and RET are part of a common signaling pathway that promoters ureteric bud proliferation and branching. This pathway may not only be critical for early kidney development and growth, but may also function in aberrant proliferation of ureteric bud derived cells, such as in cystic diseases of the collecting duct. The specific aims of this proposal will address the mechanism of RET signaling in a tissue culture model system. We have established a biological assay for RET activity in transformed MDCK renal epithelial cells. These cells show increased scattering, cell motility, and morphological changes in response to RET activation. We have also identified a set of novel phospho-proteins that co-precipitate with activated RET in MDCK cells. We will determine critical residues in the cytoplasmic domain of RET that are required for the biological response of MDCK cells and the binding sites for these potential second messengers using a panel of RET mutants. The RET binding proteins will be cloned by either an immunoaffinity strategy or with the yeast two-hybrid system. Furthermore, we will characterize the expression patterns of candidate second messengers and assess their roles in the developing kidney. These studies will directly address the mechanism of RET signaling and the biological responses of renal epithelial cells to activated RET.